Rheumatology Research Center, Tehran University of Medical Sciences, Tehran, Iran.
Department of Medical Immunology, School of Medicine, Tehran University of Medical Sciences (TUMS), Tehran, Iran.
Rheumatol Int. 2018 Mar;38(3):489-498. doi: 10.1007/s00296-017-3915-y. Epub 2018 Jan 22.
Systemic sclerosis (SSc), an autoimmune disease of connective tissue, is characterized by inflammation, fibrosis, and vessel endothelial damage. Products of Integrin subunit beta 2 (ITGB2) and selectin L (SELL) genes participate in several functional pathways of immune system. The aim of this investigation was to survey the transcript level of ITGB2 and SELL genes as well as methylation status of CpG sites in promoter region of differently expressed gene in PBMCs of SSc patients. PBMCs were isolated from whole blood of 50 SSc patients and 30 healthy controls. Total RNA and DNA contents of PBMCs were extracted. Gene expression was analyzed by real-time PCR using the SYBR Green PCR Master Mix. To investigate the methylation status of CpG sites, DNA samples were treated by bisulfite, amplified through nested PCR, and sequenced through Sanger difficult sequencing method. ITGB2 gene in PBMCs of SSc patients was overexpressed significantly in comparison to healthy controls. However, no altered SELL expression was observed. Three CpG sites of 12, 13 and 14 were significantly hypomethylated in patients group, despite overall methylation status of ITGB2 gene promoter revealed no significant difference between study groups. There was no statistically significant correlation between methylation status of ITGB2 promoter and the gene expression in patients. Regarding to lack of correlation of increased expression of ITGB2 with its promoter hypomethylation in SSc patients, our study suggests that upregulation of ITGB2 in PBMCs from SSc patients is probably due to another mechanism other than methylation alteration.
系统性硬化症（SSc）是一种自身免疫性结缔组织疾病，其特征为炎症、纤维化和血管内皮损伤。整合素亚基β 2（ITGB2）和选择素 L（SELL）基因的产物参与免疫系统的几个功能途径。本研究旨在调查 ITGB2 和 SELL 基因的转录水平以及 SS 患者 PBMC 中差异表达基因启动子区 CpG 位点的甲基化状态。从 50 名 SSc 患者和 30 名健康对照者的全血中分离 PBMC，提取总 RNA 和 DNA 含量。使用 SYBR Green PCR 主混合物通过实时 PCR 分析基因表达。为了研究 CpG 位点的甲基化状态，用亚硫酸氢盐处理 DNA 样本，通过嵌套 PCR 扩增，并通过 Sanger 困难测序方法进行测序。与健康对照组相比，SSc 患者 PBMC 中的 ITGB2 基因明显过表达。然而，未观察到 SELL 表达改变。尽管 ITGB2 基因启动子的总体甲基化状态在研究组之间没有显著差异，但患者组中 12、13 和 14 号的三个 CpG 位点明显低甲基化。ITGB2 启动子的甲基化状态与患者的基因表达之间没有统计学上的显著相关性。由于 SSc 患者 ITGB2 表达增加与启动子低甲基化之间缺乏相关性，我们的研究表明，SSc 患者 PBMC 中 ITGB2 的上调可能是由于除甲基化改变以外的另一种机制。
